Dendritic cell nodes

ABSTRACT

The present invention features dendritic cell nodes that can be used to vaccinate subjects against pathogens and to modulate a subject&#39;s immune system to treat or prevent various diseases and conditions.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of priority from Provisional ApplicationSer. No. 60/365,324, filed Mar. 18, 2002, which is herein incorporatedby reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Contract No.DAMD17-02-C-0130, awarded by the Defense Advanced Research ProjectsAgency (DARPA). The government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to engineered dendritic cell nodes(DCN) that can be used to vaccinate subjects against pathogens andtumors and to modulate a subject's immune system to treat or preventvarious diseases and conditions.

BACKGROUND OF THE INVENTION

Dendritic cells (DCs) are involved in the initiation of both innate andadaptive immune responses. These “professional” antigen-presenting cellsact cellular sentinels in every tissue of the human body, by detectingforeign antigens that serve as molecular signals of pathogen invasion.

During the adaptive immune response, an immature DC engulfs an antigen(e.g., an antigen from a pathogen, tumor, infected cell or otherabnormal cell, or a self-antigen), after which the DC undergoes amaturation process and migrates to a lymph node. Over the course of thismaturation process, the foreign antigen is cleaved into small peptideswithin the dendritic cell. These peptides are bound to majorhistocompatibility complex (MHC) class I and II molecules and presentedon the surface of the mature dendritic cell. By presenting suchprocessed peptides to T cells and B cells within the lymph node, maturedendritic cells directly and indirectly activate various subsets ofthese and other cells of the immune system, thereby guiding a series ofimmune responses that ultimately lead to elimination of pathogens.

Dendritic cells are not only critical for the induction of immuneresponses; they are also known to be important in the development ofimmune tolerance (e.g., to “self” antigens); when this process goesawry, autoimmune disease can result.

Infectious agents and tumor can evade endogenous dendritic cellsurveillance through various mechanisms. To overcome these endogenousevasion mechanisms, therapies involving the injection of dendritic cellsthat have been stimulated with specific antigens ex vivo are beingdeveloped. For example, injections of antigen-stimulated dendritic cellshave proven effective in animal models as both protective andtherapeutic cancer vaccines. However, the first trials of dendriticcells therapy in humans have shown efficacy in only a small number ofpatients. In particular, it has been found that most of the injecteddendritic cells die rapidly and fail to reach lymph nodes, andtherefore, do not succeed in activating downstream T-cell and B-cells.

Accordingly, there is a need in the art for improved dendritic celltherapies.

SUMMARY OF THE INVENTION

The present invention provides bioengineered dendritic cell nodes thatcan be used to modulate a subject's immune system. For example, thebioengineered dendritic cell nodes of the invention can be used tovaccinate a subject against one or more pathogens, to stimulate asubject's immune system against a tumor antigen for the treatment orprevention of cancer, or to tolerize a subject to an antigen (e.g., totreat or prevent allergies, asthma, autoimmune diseases, and rejectionof transplanted cells, tissues, or organs).

In a first aspect, the invention features a dendritic cell nodecomprising a biocompatible scaffold material, a chemokine for attractingimmature dendritic cells, a chosen antigen, and a maturation signal fordendritic cells.

In a second aspect, the invention features a dendritic cell nodecomprising a biocompatible scaffold material, a chemokine for attractingmonocytes, a factor that induces differentiation of monocytes intoimmature dendritic cells, a chosen antigen, and a maturation signal fordendritic cells.

In a third aspect, the invention features a dendritic cell nodecomprising a first layer for attracting immature dendritic cells intothe dendritic cell node, a second layer for presenting a chosen antigento the immature dendritic cells, and a third layer for attractingdendritic cells and inducing maturation of dendritic cells.

In a fourth aspect, the invention features a dendritic cell nodecomprising a first layer for attracting immature dendritic cells intothe dendritic cell node and for presenting a chosen antigen to theimmature dendritic cells, and a second layer for attracting dendriticcells and inducing maturation of dendritic cells.

In a fifth aspect, the invention features a dendritic cell nodecomprising a first layer for attracting monocytes into the dendriticcell node, a second layer for inducing differentiation of the monocytesinto immature dendritic cells, a third layer for presenting a chosenantigen to the immature dendritic cells, and a fourth layer forattracting dendritic cells and inducing maturation of dendritic cells.

In a sixth aspect, the invention features a dendritic cell nodecomprising a first layer for attracting monocytes into the dendriticcell node and for inducing differentiation of the monocytes intoimmature dendritic cells, a second layer for presenting a chosen antigento the immature dendritic cells, and a third layer for attractingdendritic cells and inducing maturation of the dendritic cells.

The dendritic cell node of any of the above aspects of the invention canoptionally comprise a symmetry layer. For example, the symmetry layercan be a second antigen presentation layer.

The dendritic cell node of any of the above aspects of the invention canoptionally comprise a biocompatible encapsulating layer. For example,the encapsulating layer can be biodegradable, and can contain at leastone bioactive substance to be released via diffusion from theencapsulating layer or via degradation of the encapsulating layer.

The antigen carried by the dendritic cell node of any of the aboveaspects of the invention can be a polypeptide, a peptide, a DNAmolecule, or an RNA molecule.

In any of the above aspects of the invention, the dendritic cell nodecan optionally comprise cells. The cells can be autologous ornon-autologous cells (e.g., but not limited to, monocytes or immaturedendritic cells), which can be introduced ex vivo or in vivo. Immaturedendritic cells can optionally be pulsed with antigen prior to beingintroduced into the dendritic cell node.

The dendritic cell node of any of the above aspects of the invention canbe a folded construct, e.g., but not limited to, a four-quadrant foldedconstruct. Alternatively, the dendritic cell node of any of the aboveaspects of the invention can be a rolled construct.

At least one layer of the dendritic cell node of any of the aboveaspects of the invention can comprise a polymer for sustained release ofa factor embedded within the polymer. In one example, the factor can bewithin microspheres or nanoparticles, wherein the microspheres ornanoparticles are embedded within the polymer and undergo sustainedrelease from the polymer.

The dendritic cell node of any of the above aspects of the invention cancomprise at least one layer comprising bioconcrete, wherein thebioconcrete comprises a biodegradable mesh piercing a polymer gel.

In a seventh aspect, the invention features a method of constructing adendritic cell node as described in any of the first six aspects of theinvention. The method includes the steps of: a) depositing a first layeronto a substrate, and b) depositing each successive layer onto aproceeding layer, thereby constructing the dendritic cell node. Any ofthe dendritic cell nodes of the invention can be constructed in thesequential order of first layer to last layer, or in the reverse order,i.e., last layer to first layer.

For example, in an eighth aspect, the invention features a method ofconstructing a dendritic cell node. The method includes the steps of: a)depositing, onto a substrate, a layer for attracting immature dendriticcells into the dendritic cell node; b) depositing, onto layer (a), alayer for presenting a chosen antigen to the immature dendritic cells;and c) depositing, onto layer (b), a layer for attracting immaturedendritic cells and inducing maturation of the immature dendritic cells.Alternatively, the method can include the steps of: d) depositing, ontoa substrate, a layer for attracting immature dendritic cells andinducing maturation of the immature dendritic cells; e) depositing, ontolayer (d), a layer for presenting a chosen antigen to the immaturedendritic cells; and f) depositing, onto layer (e), a layer forattracting immature dendritic cells into the dendritic cell node,thereby constructing an dendritic cell node.

In a ninth aspect, the invention features a method of constructing adendritic cell node including: a) depositing, onto a substrate, a layerfor attracting monocytes into the dendritic cell node; b) depositing,onto layer (a), a layer for inducing differentiation of the monocytesinto immature dendritic cells; c) depositing, onto layer (b), a layerfor presenting a chosen antigen to immature dendritic cells; d)depositing, onto layer (c), a layer for attracting dendritic cells andinducing maturation of dendritic cells, thereby constructing a dendriticcell node.

The ninth aspect of the invention can further include the step of: e)depositing, onto layer (d), a layer for presenting a chosen antigen toimmature dendritic cells, such that the dendritic cell node comprisestwo layers for presenting a chosen antigen to immature dendritic cells.

In a tenth aspect, the invention features a method of stimulating animmune response in a subject, comprising administering, to the subject,a dendritic cell node as described in any of the above aspects of theinvention, wherein the dendritic cell node comprises an antigen and adendritic cell maturation factor sufficient to stimulate an immuneresponse against the antigen, thereby stimulating the immune response inthe subject. The antigen can be e.g., from an infectious agent (e.g., avirus, a gram-negative bacterium, a gram-positive bacterium, a fungus, aprotozoan, a rickettsium) or e.g., from a tumor cell.

In an eleventh aspect, the invention features a method of inhibiting animmune response in a subject, comprising administering, to the subject,a dendritic cell node as described in any of the above aspects of theinvention, wherein the dendritic cell node comprises an antigen and adendritic cell maturation factor sufficient to inhibit an immuneresponse against the antigen, thereby inhibiting the immune response inthe subject. For example, the antigen can be an allergen, a self-antigen(e.g., in autoimmune disease), or a non-self-antigen (e.g. on anon-autologous transplanted cell, tissue, or organ).

In a twelfth aspect, the invention features a method of attractingimmature dendritic cells to a specific location within the body of asubject, comprising administering, to the subject, the dendritic cellnode of the first, third, or fourth aspect of the invention.

In a thirteenth aspect, the invention features a method of attractingmonocytes to a specific location within the body of a subject,comprising administering, to the subject, the dendritic cell node of thesecond, fifth, or sixth aspect of the invention.

In a fourteenth aspect, the invention features a method of slowingbiodegradation of a polymer gel, comprising enclosing the polymer gelwithin a biodegradable mesh structure, thereby slowing biodegradation ofthe polymer gel. The polymer gel can contain a bioactive substance, inwhich case, the method slows release of the bioactive substance from thepolymer gel. Moreover, the biodegradable mesh can optionally contain abioactive substance to be released via diffusion from the biodegradablemesh or via degradation of the biodegradable mesh.

In a fifteenth aspect, the invention features bioconcrete, comprising apolymer gel carried within a biodegradable mesh. In one example, thebioconcrete can contain a bioactive substance within the polymer gel. Inanother example, the bioconcrete can contain a bioactive substancewithin the biodegradable mesh, wherein the bioactive substance isreleased via diffusion from the biodegradable mesh or via degradation ofthe biodegradable mesh.

In a sixteenth aspect, the invention features a method of preparing anantigen for uptake by a dendritic cell, comprising encapsulating theantigen within nanoparticles or microspheres, thereby preparing theantigen for uptake by a dendritic cell.

In a seventeenth aspect, the invention features a method of enhancinguptake of an antigen by a dendritic cell, comprising delivering theantigen packaged within nanoparticles or microspheres to the dendriticcell, thereby enhancing uptake of the antigen by the dendritic cell.

In any of the above aspects of the invention, the antigen can be apolypeptide, a peptide, a DNA molecule, or an RNA molecule. The antigencan also be a library of polypeptides, peptides, DNA molecules, or RNAmolecules.

Additional advantages of the invention will be set forth in part in thedescription which follows, and those skilled in the art will recognizethat other and further changes and modifications may be made theretowithout departing from the spirit of the invention. The advantages ofthe invention will be realized and attained by means of the elements andcombinations particularly pointed out in the appended claims. It is tobe understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the architecture and various layers andcomponents of an exemplary DCN.

FIG. 2(a)-(b) show, respectively, a photograph and a drawing of thebiological architecture tool (BAT).

FIG. 3 is a diagram showing the chemical composition of hyaluronic acid.

FIG. 4 is a depiction of two photographs showing a pyramid-shaped,collagen/gelatin engineered tissue construct (ETC) containing eightlayers.

FIG. 5 is a depiction of two photographs displaying a vehicle (leftpanel) and capsule (right panel) built with PF-127/PPF-PEG mix.

FIG. 6(a)-(g) is a depiction of a series of photographs showing: (a)layer-by-layer construction of a capsule; (b) filling the capsule withvarious layers of the DCN; (c) a filled capsule; (d) rinsing the filledcapsule in saline and cutting it off the slide; (e) fitting the filledcapsule into an injection needle; (f) close view of capsule in needle;(g) subcutaneous injection of capsule into a chicken.

FIG. 7(a)-(b) is a depiction of two photographs showing mesh formsfabricated by the BAT; (a) shows a two-layer PPF “log cabin”; (b) showsa four-layer PCL mesh.

FIG. 8(a)-(c) is a depiction of three photographs showing a viabilitytest in a test-well constructed using the BAT and the compositions andmethods of the invention. (a) shows a PF-127/PPF-PEG test-well filledwith fibrin glue; (b) shows fibroblasts deposited together with thrombininto the test-well; (c) shows the fibroblasts after a 48-hour incubationat 37° C.

FIG. 9 is a diagram showing three strategies for controlled release fromthe DCN: (1) cross-linked networks; (2) controlled release microspheres;and (3) controlled release nanoparticles.

FIG. 10(a)-(b) is a pair of graphs showing controlled release ofproteins from: (a) triblock hydrogels encapsulating bovine serumalbumin; and (b) PLGA/PEG microspheres encapsulating ovalbumin.

FIG. 11(a)-(b) respectively show: (a) an NMR spectrum showing thestructure of a PGLA-PEG-PLGA triblock copolymer (arrows and shadingindicate the corresponding resonances from the schematic structure); and(b) a graph showing the results of a triblock hydrogel toxicity assay(100 mg of PGLA-PEG-PLGA was photo-polymerized in one culture well; onDay 7, bone marrow-derived dendritic cells were added to the well withthe gel (solid bars) or to the controls (open bars) and were culturedfor 24 hours). FIG. 12 is a series of panels relating to drug deliverycomponents: (a) is a depiction of an optical micrograph (OM) showingprotein-loaded PLGA microspheres; (b) is a schematic ofPLGA-PEG-PLGA-based hydrogel nanoparticles; (c) is a depiction of ascanning electron micrograph (SEM) showing nanoparticles; (d) is adepiction of an ethidium bromide-stained gel showing DNA recovered frombiodegradable nanoparticles lysed with 0.1 M NaOH; (e) is a depiction ofa pair of photomicrographs (left=brightfield, right=fluorescence) ofdendritic cells containing phagocytosed nanoparticles.

FIG. 13 is a chart showing various factors to consider when choosingbiomaterials for the dendritic cell node.

FIG. 14 is a representation of a photomicrograph showing fMLP dropletsclose-up on a scaffold patch.

FIG. 15 is a representation of a photomicrograph of fMLP dropletsdeposited on a scaffold patch, which shows that the scaffold margins arefree of droplets.

FIG. 16 is a depiction of a pair of photomicrographs showing triblockgel particle uptake by dendritic cells after two hours in culture(left=bright field; right=fluorescence).

FIG. 17 is a depiction of an ethidium bromide-stained gel showing DNAencapsulation in degradable nanogel particles.

FIG. 18 is a graph showing attraction of immature dendritic cells tofMLP peptide.

FIG. 19 is a depiction of the results of a microarray analysis showinggene expression in human monocyte-derived dendritic cells.

FIG. 20 is a graph showing a strategy for producing a dendritic cellnode with a folded quadrant structure.

FIG. 21 is a diagram showing a strategy for producing a dendritic cellnode with rolled layers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides dendritic cell nodes (DCN) and methodsfor making and using the same. The DCN, as described herein, is animplantable, three-dimensional (3D), tissue-engineered (TE) scaffoldthat can be used to modulate (increase or decrease) the immune responsesof a subject. Accordingly, the DCN can be used to stimulate the immunesystem, e.g., to vaccinate against infectious agents or to treat orprevent cancer. The DCN can also be used to tolerize against antigens,e.g., to treat or prevent allergies, asthma, autoimmune disease, orrejection of transplanted organs, tissues, or cells.

The DCN is an engineered tissue construct (ETC) that contains basescaffold materials and biomolecules. The term “base scaffold materials”refers to the biomaterials used to construct the ETC, such as (but notlimited to) collagen, fibrin glue, hyaluronic acid (HA), triblockcopolymers, poly(lactide-co-glycolide) (PLGA). Biomolecules include,e.g., chemicals, vitamins, hormones, molecules, proteins, nucleic acidmolecules (e.g., plasmid or viral vectors), antigens, chemokines, andcytokines, that are located within the base scaffold material to inducea specific response and/or functionality. In addition, the DCN canoptionally be populated with cells during its fabrication.

Abbreviations and symbols used throughout this specification are setforth in Table 1.

Dendritic Cells

The human body's immune system is a complex and potent network, theadaptability of which is mediated by several key cell types, the mostimportant of which are dendritic, T, and B cells. Toll-like receptors(Tlr) are believed to be the first line of recognition at the time ofpathogen encounter (Takeda K, Kaisho T, Akira S. Toll-like receptors,Annu Rev Immunol. 2003; 21:335-76). DC's, which are the most potentantigen-presenting cells (APC's) known, express a large number of theten known Tlr genes and can be used to develop novel TE vaccines.

DC's serve as cellular sentinels, standing guard in every tissue of thehuman body, ready to detect the antigens that are the molecular signs ofpathogen invasion. DC's initiate both adaptive and innate immuneresponses (Ref. 1). They are the most powerful APC type; they ingestantigens at infection sites and present them in lymphoid organs to Tcells as peptides bound to both Major Histocompatibility Complex (MHC)class I and II products. DC's initiate and control the quality of theT-cell response, driving the transformation of naïve lymphocytes intodistinct classes of antigen-specific effector cells. In addition, DCsdirectly stimulate the adaptive B cell responses (Litinskiy M B,Nardelli B, Hilbert D M, He B, Schaffer A, Casali P, Cerutti A. DCsinduce CD40-independent immunoglobulin class switching through BLyS andAPRIL. Nat Immunol. 2002 September; 3(9):822-9; Craxton A, Magaletti D,Ryan E J, Clark E A. Macrophage- and dendritic cell-dependent regulationof human B-cell proliferation requires the TNF family ligand BAFF.Blood. 2003 Jan. 16 12531790; MacLennan I, Vinuesa C. Dendritic cells,BAFF, and APRIL: innate players in adaptive antibody responses.Immunity. 2002 September; 17(3):235-8; Schneider P, MacKay F, Steiner V,Hofmann K, Bodmer J L, Holler N, Ambrose C, Lawton P, Bixler S,Acha-Orbea H, Valmori D, Romero P, Werner-Favre C, Zubler R H, BrowningJ L, Tschopp J. BAFF, a novel ligand of the tumor necrosis factorfamily, stimulates B cell growth. J Exp Med. 1999 Jun. 7;189(11):1747-56.) DC's are also critical players in innate immunity.They produce cytokines important to host defense and to activation ofnatural killer cells (NKC's) that kill target cells and produceimportant cytokines (Ref. 2).

Before leaving the lymph node, T cells also activate B cells (in synergywith the indirect and direct effects of dendritic cells on B cells),which then produce antibodies that bind to pathogens or to their toxicproducts and prevent their harmful effects. Dendritic, T, and B cellsalso recruit other classes of immune cells to participate in thwartingan invading pathogen. Effectively, DC's trigger and guide a chainreaction of immune responses that leads to elimination of a pathogen.

Described herein are bioengineered, DC-activating ETC's, containing DC'sor not, that transmit molecular signals to activate the body's DC's,which can be released and then typically either migrate to the naturalhost lymph nodes; or mature and entice T cells to enter and triggerfurther immune responses at the site of vaccination. The two generalapproaches to DCN construction are as described in Table II.

In a first example, TE scaffolds are not populated with DC's duringfabrication, but are endowed with (a) chemokines that attract immatureDC's (iDC's) or monocytes; (b) the pathogenic antigen(s); (c) various DCmodulators, as will be discussed later for immunity; and/or (d);suppressors for immune tolerance to induce mature DC's to migrate fromthe DCN to “natural/host” draining lymph nodes after programming andantigen-loading has occurred.

This DCN embodiment is an implantable DC docking vaccine; this type ofDCN includes the ability to concentrate a large number of DC's in asmall area subcutaneously. These DCNs can include appropriate antigensfor the pathogen, for example, using recombinant proteins or peptides(or libraries thereof), DNA molecules (e.g., plasmids, viral vectors,etc.) or RNA molecules that encode the desired antigen (or librariesthereof), and appropriate state inducers to program the optimal responsefor a pathogen and to induce DC's to migrate from the DCN to“natural/host” draining lymph nodes after antigen loading andprogramming has occurred.

A porous ETC is created that can release factors with finecontrol—concentration and start/end times using biodegradablemicrospheres or by appropriately embedding the biomolecular factors inthe scaffold host material—in the same way that the body does during aresponse.

In a second example, ETC's can be populated with DC's duringfabrication. Controlled exposure to signaling molecules (e.g., cytokinesand chemokines) together with engineered antigens (based on pathogens'molecular components) in an ETC allow optimal activation of DC's so thata powerful immune response is initiated. For either type of DCN(fabricated with or without DC's), afterwards, these constructs aresubcutaneously injected into the patient prior to tumor and/or pathogenchallenge. The best scaffold, microenvironment, gradients, andconcentrations are optimized, all of which are provided by the tools andmethods disclosed herein. Table III provides examples of ligands for usein modulation of DC's on the scaffold.

In vivo attraction and repulsion of DC's has been shown by thesuccessful attraction of iDC's to subcutaneously implanted polymer rods(Ref. 3). These DC's were loaded with a tumor-associated antigen andnaturally emigrated, repelled from the rods and were found to home tolymph nodes (Ref. 4). The 3D scaffolds described herein not only allowthe attraction and repulsion of DC's, but also the selection for optimalDC subtypes and the modulation of their maturation state to maximize theefficiency of antigen presentation to the immune system.

Effective DC-based immunotherapies are developed through the rationalmanipulation of DC's with scaffolds and deposition, and, variousmodulators to maintain their proper activation and maturation states,enhance their viability, and facilitate their migration to lymph nodes.Disclosed are artificial TE dendritic cell nodes that can be repackagedfor cures for diabetes, arthritis, lupus, cancer, infectious disease,autoimmune diseases (such as Type I Diabetes, Lupus, rheumatoidarthritis, multiple sclerosis and others). The DCN can be redesigned totarget one disease at a time by controlling the maturation states of theDC's and/or loading them with the proper antigen(s) associated with thetarget antigen of interest. Furthermore, the DCN can also develop a TEscaffold for inducing tolerance, because the DC is involved intolerance. It is then possible to address a vast number of inflammatorydiseases, including autoimmunity, allergy, and asthma.

Dendritic Cell Properties

As mentioned above, DC's protect human tissues by detecting the antigensthat are the molecular signs of pathogen invasion. DC's are APC's with aunique ability to induce primary immune responses. DC's capture andtransfer information from the outside world to the cells of the adaptiveimmune system. DC's can initiate both adaptive and innate immuneresponses (Ref. 5). DC's are not only critical for the induction ofprimary immune responses, but may also be important for the induction ofimmunological tolerance, as well as for the regulation of the type ofT-cell-mediated immune response.

DC's initiate an immune response in various ways. Immature DC's candirectly interact with pathogens that induce the secretion of cytokines.e.g., interferons (IFN's), which in turn can activate the immune system.After capturing antigens, iDC's migrate to lymphoid organs (e.g., lymphnodes) where they mature. After maturation, they display peptide MHC's,thereby enabling the selection of rare circulating antigen-specificlymphocytes. Thus, DC's initiate and control the quality of the T-cellresponse, driving the transformation of naïve lymphocytes into distinctclasses of antigen-specific effector cells. Activated T cells are ableto migrate and reach the diseased tissue. Helper T cells (CD4⁺ T cells,Type I; symbol T_(H)1) secrete cytokines, which permit activation ofmacrophages, NKC's, and cytotoxic CD8⁺ T cells. Cytotoxic T cellseventually lyse (kill) the diseased or infected cells. Specifically,CD8⁺ T cells directly kill the tumor or pathogen. Other T-helpers (ofType II; symbol T_(H)2) activate B cells, which produce antibodies thatbind to pathogens or to their toxic products, thereby preventing theiraccess to cells. Using the cytokine network, dendritic, T, and B cellsalso recruit other classes of immune cells to participate in thwartingan invading pathogen. Effectively, DC's trigger and guide a chainreaction of immune responses that leads to elimination of a pathogen.

From the aforementioned chain of events, it has been hypothesized thatDC's are a link between innate immunity and adaptive immunity inantitumor immune responses (Ref. 6-7).

Immune Response Evasion Mechanisms

Even though DC's are a key component of immunological strategies,infectious agents and tumors can evade DC surveillance through severalmechanisms. Certain agents may not produce inflammation, which normallyfacilitates antigen uptake by DC's. Some microorganisms might restrainDC's by producing inhibitory molecules (Ref. 8). To address theseevasive mechanisms, therapies based on the injection of DC's, chargedwith antigens ex vivo, are being actively developed.

Dendritic Cell Therapy

In the field of cancer treatment, DC-based treatments have demonstratedregression of tumors. Tumor-specific antigens are presented to DC's incontrolled conditions outside the body; these antigen-loaded DC's arethen injected to initiate an immune response. In animal models, DCtherapy has proven effective both as cancer vaccines and immunotherapy.Injection of bone-marrow-derived DC's pre-pulsed with tumor-associatedpeptides has been shown to protect mice against subsequent lethal tumorchallenge (Ref. 9). Moreover, in mice bearing established macroscopictumors, treatment with tumor-peptide-pulsed DC's resulted in sustainedtumor regression and tumor-free status in 80-100% of cases (Ref. 9-10).Similar results have been observed with the injection of tumorlysate-pulsed DC's in mice (Ref. 7). The injection of DC's charged withtumor-associated antigens (Ref. 9-11) has proven effective in animalmodels both as protective cancer vaccines and as therapies to eliminatepreexisting tumors.

Dendritic Cell Vaccination Results in Humans

Injections of DC's charged with antigens (Ref. 9-11) have proven veryeffective in animal models as both protective and therapeutic vaccinesas discussed above. However, the first trials of DC therapy in humanshave only shown efficacy in a small number of patients (Ref. 12-13).Whereas numerous factors might be involved in the treatment's lowefficacy, a consistent finding has been that most of the DC's died uponinjection. Because of improper maturation, very few (0.1%) DC's reachedthe natural lymph nodes. Improvement of this therapy has recently beendemonstrated in animal studies when DC viability, activity, and stateare enhanced by turning on certain genes in DC's by modulators (Ref.14-15). In addition, recent human trials with DC vaccination forinfluenza have clearly demonstrated the importance of the DC activationand maturation states in eliciting potent responses (Ref 3, 16).

Why Use Engineered Constructs?

The present invention provides TE scaffolds as a means to overcomespecifically the aforementioned obstacles in DC-based vaccines. TEscaffolds provide the following attributes as they pertain towards theDCN for vaccine discovery:

Scaffolds endowed with appropriate biomolecules (cytokines) will help toextend the life of the DC's and to activate and mature themappropriately, thus enabling a more potent effect with fewer injections.

Targeted antigens for presentation by DC's are controlled by TEscaffolds.

State modulators of the DC's are controlled by incorporation of theseligands in the TE scaffold.

Dendritic Cell Node Overview

The DCN is an ETC that can be introduced (e.g., subcutaneously) into ahuman or other animal. The DCN contains various chemoattractant layersthat, variously: (1) attract endogenous monocytes (or other DCprecursors) from the host animal in which the DCN is implanted, (2)induce differentiation of the host monocytes into immature DC's, (3)load the immature DC's with specific antigens, and (4) induce maturationof DC's, which then migrate to a draining host lymph node. At theendogenous host lymph node, the mature DC's activate endogenouspre-programmed naive T and B cells (the ones matched for the antigenfrom the large repertoire of T and B cells). The natural host lymph nodeis the location where of T and B cells reside and find their matchedantigen.

The DCN, as shown in FIG. 1, has the abilities to: (1) differentiatemonocytes 0105 to iDC's 0135; (2) attract both monocytes 0105 and DC's0135 and 0155 alike via chemotactic layers; (3) load antigens 0132 ontothe iDC's 0135; and (4) differentiate these iDC's 0135 into mature DC's0155 both in vitro and in vivo. Various different antigens 0132associated with a number of diseases, e.g., (but not limited to) cancer,diabetes, human immunodeficiency virus (HIV), malaria, can be used.Other permutations to achieve the DCN functionality are also possible.For example, the DCN can be constructed in such a way that functions ofseveral of the layers are combined; only three layers are necessary,with the three layers being an antigen-presenting layer, a maturationsignal layer with appropriate ligands, and an antigen-presenting layerwith a DC chemokine in all three layers. In this case, the monocyterecruitment layer 0110 and/or the differentiation layer 0120 is notincluded, as the DCN simply attracts DC's already in the body.

To build biocompatible structures that replicate or enhance the naturalliving system (microenvironment, 3D structure, chemotactic gradients,etc.) to support cell development, the disciplines of digitalmanufacturing, tissue engineering, and immunology are incorporated tocreate the DCN. The digital printingcomputer-aided-design/computer-aided-manufacturing (CAD/CAM) techniquesof the Biological Architectural Tool (BAT) are used to build designer 3Dheterogeneous ETC's; however, in principle, other digital printing toolsmay also be used.

The BAT is a 3D, multiple-head, through-nozzle printing machine, shownin FIG. 2, which can be used to directly deposit the components of theDCN, such as biomaterials, cells, and molecular cofactors (the BAT isdescribed in detail in PCT/US02/26866, herein incorporated by referencein its entirety for its teachings regarding how to make and use theBAT). Examples of such biomaterials, cells, and molecular cofactorsinclude, but are not limited to:

Biomaterials: collagen, ECM materials, fibrinogen, thrombin, fibringlue, HA, PLGA, PPF-PEG, PCL, gelatins (including photocurablegelatins), Pluronic F-127, triblock A-B-A (e.g., PLGA-PEG-PLGAdimethacrylate) copolymers.

Cells: endothelial, epithelial, dendritic, T, and B cells; monocytes,macrophages, neurons, fibroblasts, stem cells.

Molecular Cofactors: cytokines, chemokines, DNA plasmids, libraries ofexpressed antigens, proteins, glycoproteins, peptides, vitamins.

These materials are deposited onto various supporting substrates andsurfaces to create surrogate tissues and experimental platforms forexperiments in cell biology and tissue engineering. The BAT deposits theDCN and other ETC's in a layer-by-layer (LBL) mode. The device (FIG.2(a)) consists of an xyz coordinate stage 0200; a number ofmicrodispensing deposition heads or pens 0210, each of which has anindividual observation and tuning video camera 0220; a light source tocure photopolymers in-line 0230; a system of individual temperaturecontrol for the pens and the stage 0240; compressed air to pressurizepens 0250; a humidifier preventing dehydration of living samples; and acomputer controlling the whole deposition process (the latter two notshown). The BAT has been designed as an upgradeable system, allowingmore units and functions to face upcoming tasks to be built therein.

Base Scaffold Materials Used to Fabricate the DCN

Next are discussed candidate base scaffold materials for constructingthe DCN in LBL mode using a digital printing apparatus, i.e., thebiomaterials. Later, the role and ingredients (biomolecules) of eachlayer in the entire construct is presented in detail; i.e., the variousmolecular factors that are added to each layer in the multilayer DCN.The synthetic and natural polymers (biomaterials) shown in Table IV areonly representative. Other biomaterials and configurations can also beused. Below are presented several specifics regarding a few of thecandidate scaffold materials.

Base Scaffold Biomaterials

Biomaterials as set forth in Table IV can be used to construct the basescaffolds and associated capsules of the DCN. The base scaffoldbiomaterials simply need to be of good construction properties (retaintheir shapes), and be biocompatible and biodegradable, etc., as shown inFIG. 13.

Fibrin Glue

This fibrinogen-thrombin-calcium(II) system produces stable clots firmlyattached to various surfaces. This system can be combined with naturalcomponents like HA and collagen, thus providing the necessary stickinessand stability of gel layers in aqueous solutions. Several fibrin gluepatches containing laminin have been fabricated for cell viability andhave shown promising results. One particular fibrin biomaterialconfiguration is detailed below. The following description is exemplaryonly, as other combinations can be used without departing from thespirit and scope of the invention.

These fibrin glue patches were 5×5 mm squares deposited in 30-mm plasticPetri dishes, one patch per dish. The patches were deposited in LBL modeusing two different solutions: (1) Solution “Fibro” contained 80 mg/mLfibrinogen and 0.1 mg/mL laminin in distilled water; (2) Solution“Thrombo” contained 22 mg/mL thrombin in a solution containing 20 mMCaCl₂ and 1% w/w HA.

Samples:

-   A1-A6: “Fibro” deposited first, “Thrombo” second.-   B1-B2: Same as A series, except “Fibro” reduced about 30%.-   C1-C3: “Thrombo” deposited first, “Fibro” second.-   D1-D4: Same as C series; the deposition rate for “Fibro” was reduced    3× while total quantities were kept the same.    Estimated Loading of Components in the Patches:-   Laminin: 6±2 μg/cm²-   Fibrin clot: 3±1 mg/cm²-   HA: 0.40±0.15 mg/cm²

The foregoing description of fibrin glue patches is an example only anddoes not limit the concentrations of ingredients used in such patches.For example, the fibrinogen concentration can be from about 0.1 mg/ml toabout 100 mg/ml, e.g., about 0.1 to about 1 mg/ml, about 1.0 to about 10mg/ml, or about 10 to about: 20, 30, 40, 50, 60, 70, 80, or 90 mg/ml.The thrombin concentration can be about 0.1 mg/ml to about 30 mg/ml,e.g., about 0.1 to about 1 mg/ml, about 1.0 to about 10 mg/ml, or about10 to about 20 or about 30 mg/ml.

Hyaluronic Acid: A Universal Thickening Additive

Hyaluronic acid (HA) is a universal component of the extracellularspaces of body tissues. This mucopolysaccharide has an identicalchemical structure whether it is found in bacteria or human beings. Itis composed of repeating disaccharide units of N-acetylglucosamine andD-glucuronic acid as shown in FIG. 3.

HA retains significant amounts of water to form a liquid gel. HAincreases the viscosity of fluids, thus facilitating control andimproving quality of deposition for cellular suspensions as one example.

HA is miscible with any synthetic or natural material listed in Table Vwithout side effects. Being a natural component of the ECM material, itis harmless to cells. Preliminary results indicate that a 1% solution ofHA supports the suspension of cells for days, preventing earlyagglomeration. Thus, this should be an ideal biomaterial component forsuch ETC's as the DCN.

Generally, the fibrin glue and HA additives to such natural polymers ascollagen and ECM show significantly improved construction/buildingproperties, allowing the ETC to be built in LBL mode.

Collagen and Gelatin Layers

Collagen and gelatin layers also make promising scaffold materials. FIG.4 shows photographs of an alternating collagen/gelatin eight-layerpyramid construct. The gelatin has greater construction properties;however, the collagen shows improved construction upon adding fibringlue and HA to the scaffold matrix. Both the collagen, gelatin, HA, andECM natural polymers are soluble in bodily fluids and can degradequickly. Methods are disclosed below on how to decrease the degradationrate of these natural polymers using bioconcrete.

PF-127

The use of PF-127 in combination with PPF-PEG (22%-25% and 12%-10%solutions in phosphate-buffered saline (PBS), respectively) allow thebuilding of sophisticated 3D constructs, including closed boxes andcapsules stabilized by photo-crosslinking of PPF-PEG as shown in thenext section. In general, PF-127 mixed with other viscous componentsretains its remarkable shape-forming capacity, but only to a limit. Whenthe share of the other component exceeds a certain level, the solutionwill likely lose the feature of reverse-temperature gelation intrinsicto PF-127 and turn into a primitive, viscous syrup.

Injectable Capsule Made of PF-127/PPF-PEG Combination

DCN constructs comprising a number of layers of combined natural andsynthetic materials can be encapsulated in a miniature vehicle, thematerial of which can act like an antigen or cytokine depot carrier aswell. Hard gelatin, e.g., can be used for this task. The injectablecapsule can serve as a temporary “housing” for the proper DCN ETC. Thecapsule in this case is used to withstand the shear forces uponinjecting the DCN ETC in the patient via subcutaneous injection.

As one example, a combination of PF-127 with PPF-PEG provides excellent3D printing and stability in aqueous environments due tophoto-crosslinking of the PPF-PEG component. FIG. 5 shows a vehicle anda capsule built with the PF-127/PPF-PEG mixture. The box measures 5×5×2mm; the capsule is 7×1.4×0.8 mm. PF-127 has been successfully used forcontrolled subcutaneous delivery of drugs, including insulin. It couldprobably alleviate any possible negative effects of PPF-PEG on cells.

An injectable capsule represents a rectangular box 7×1.4×0.8 mm that canbe filled with fibrin glue, urinary bladder mucosa (UBM)/HA mixture,photocurable gelatins, PCL, or another biomaterial of choice “in-line,”utilizing the multiple-head BAT system. In this particular case, theinjectable capsule would be filled with the multilayer DCN ETC shown inFIG. 6(b). The capsule deposited on the glass slide can be easilydetached and inserted into a special needle for a subcutaneousinjection, as shown in FIG. 6(d)-(g).

The injection needle used in these experiments was supplied with aplastic plunger that pushed the capsule out. Injected with due care, thecapsule remained undamaged. It is envisaged that subcutaneous injectionof the DCN will be required for functionality. One of ordinary skill inthe art will understand that such vehicles for enclosing the DCNs of theinvention can be made in any convenient shape, e.g., square,rectangular, or other-shaped box, capsular, spherical, ovoid,cylindrical, etc.

Capsules

Capsules such as shown in FIG. 5 and FIG. 6 should keep all elements ofthe device together for the time necessary for curing or experimentalobservation. Meanwhile, they should allow cell migration both fromoutside into the device and vice versa, as necessary. Sensitive andeasily soluble materials like collagen-bearing signaling peptides shouldbe protected by the capsule from early erosion. In contrast, structuralelements of the capsule can and should work themselves as erodingvehicles for chemoattractants and cytokines to release them in due time.All of these properties can be attained using the “bioconcrete” and“mesh basket” concepts discussed below.

Degradable Mesh

A degradable mesh as shown in FIG. 7 is fabricated by the BAT from suchphotoreactive materials as PPF, PPF-PEG, or PPTD, or by thesolidification of viscous yet volatile solutions of PCL or PLCL. Thewire probes show the open channels in FIG. 7(a). These mesh structureswill become elements of more-complex devices.

Bioconcrete

Biodegradable mesh structures made from the relatively hard materialsnamed above can become “rebars” in composite blocks wherein the role of“cement” is assigned to soft hydrogels, either natural, such ascollagen, HA, ECM, or fibrin glue, or synthetic, such as PEG derivates.Liquid sols deposited on the top of reasonably thick mesh packs willpenetrate inside, congealing afterwards. Those composite structures willbe able to retain soft gels significantly longer than the exposed gels.Thus, the biodegradability of the natural polymers can be significantlyextended in the bioconcrete meshes. Accordingly, these reinforced gelscan serve as reliable and long-lasting depots for more-hydrophiliccytokine peptides and other bioactive substances that have a biologicalor physiological effect on cells or tissue, e.g., chemicals, vitamins,hormones, molecules, proteins, nucleic acid molecules (e.g., plasmid orviral vectors), antigens, and chemokines. In addition to its structuralrole, the “rebar” materials can be loaded with molecules (e.g.,chemoattractants, modulators, or antigens) that require slower releasekinetics compared with the molecules encapsulated in the gel (“cement”).For example, hydrophobic chemoattractants and other bioactivesubstances, such as the chemoattractant fMLP and its derivatives, can beloaded into the rebars. “Bioconcrete” structures can readily incorporatecells provided that the hydrogel “cement” is soft enough to allowcellular motility. Multivehicular systems of nano- and microspheresloaded with cytokines can be comfortably adopted by “bioconcrete”structures to produce an even more developed delivery system.

Mesh Basket

The mesh basket is a combination of the concept of the injectablecapsule with that of the multilayered mesh (FIG. 7(a)). Indeed, arectangular- or honeycomb-grid mesh can become the bottom of theencapsulating box, for which walls will be built in regular LBL fashion.

Platforms for Viability Tests

These tests were designed for assessing the viability of cells depositedinto various environments, placed onto materials chosen forencapsulation in the DCN, or performing another structural role. Thetest platforms (also referred as “test-wells”) were built in the 30-mmPetri dishes LBL as square boxes, about 4×4×0.3 mm, with the expandedfoundation, as shown in FIG. 8. Cell carriers, such as fibrin glue/HA orECM/HA composites, were placed in the box with cells either depositedsimultaneously or on the top of the whole construct. The medium wascarefully poured into the Petri dish to cover the construct.

PPTD and PF-127/PPF-PEG were both used to build the test-wells.Gamma-irradiated nondividing fibroblasts were used as a test culture.The construct has demonstrated viability within 48 hours at 37° C.

Microparticle Controlled Release Strategies

Having presented the base construction scaffold materials, controlledbiomolecule release strategies for the DCN are now addressed. Typicalsynthetic or natural scaffolds capable of multiple molecular-factordelivery can be fabricated from the DCN construction materials shown inFIG. 9. The resulting construct allows sustained biomolecule deliveryand maintenance of the biological activity of incorporated and releasedcytokines, chemokines, antigens, DNA plasmids, peptides, etc. Thesebiomolecules can be incorporated into scaffolds by several approaches asschematically illustrated in FIG. 9. There are generally three distincttypes of release matrices: (1) printable biomaterials (e.g., triblockcopolymer hydrogels) for the tailored release of proteins; (2)gel-immobilized degradable microspheres for the tailored release ofpeptides and small-molecule factors; and (3) gel-immobilized hydrogelnanoparticles for the tailored delivery of such biomolecules as plasmidDNA.

The first methodology involves simply mixing the biomolecules with thebase scaffold material and results in a more rapid release, e.g., hoursto weeks, as shown in FIG. 10. The base scaffold materials(biomaterials) also provide a matrix for immobilization of microspheres(e.g., PLGA/PEG) and hydrogel nanoparticles within layers of the DCN. Asone example, printable aqueous solutions have been developed of themethacrylated PLGA-PEG-PLGA triblock copolymer. These are solidified insitu during printing for either immobilization of microspheres andnanoparticles in desired locations within a specific DCN layer or fordirect encapsulation of biomolecular factors within the DCN layer. Thetriblock copolymer can be printed as a viscous aqueous solution andcured by ultraviolet photopolymerization during printing. Factors may beadded to the triblock solution and encapsulated in the hydrogel forcontrolled release (FIG. 10(a)), or the hydrogel can be used toimmobilize PLGA/PEG microspheres or triblock copolymer nanoparticles ina desired location in printed devices. For example, by blendingdifferent amounts of the hydrophilic polymer PEG with the morehydrophobic PLGA, release profiles for proteins and peptides from thesemicrospheres can be tailored, as shown in FIG. 10(b). Even though aspecific example is provided above on how to tailor the release ofproteins from PLGA/PEG nano/microspheres, the general methodology issimilar in concept for other biomaterial systems as well.

To boost the mechanical strength of natural- or biopolymer-basedscaffolds, as well as to provide materials for building biodegradablecontrolled-release components of the drug delivery devices describedherein, triblock copolymers composed of a central PEG block with shortterminal PLGA blocks were developed. As shown in the nuclear magneticresonance (NMR) data in FIG. 11(a), these are end-capped withmethacrylate or acrylate double bonds, allowing polymerization of thesematerials into a network hydrogel. Variation of the relative lengths ofthe PLGA and PEG blocks allows the degradation rate of the hydrogel tobe tuned over a broad range and release of encapsulated factors to occurover a few days or up to a month.

Hydrogels of the triblock copolymer are ideal for controlled release ofthe chemotactic proteins, since these matrices can be formed under mildaqueous conditions (room-temperature photo-polymerization) andencapsulate high concentrations of the protein in a local site in thescaffold. Degradation of the gel will control release of the proteinover time. Printing of the triblock copolymer has been tested using theBAT and it was found that it could be readily printed into 3Dconstructs. Toxicity of these materials towards dendritic cells wastested in vitro, as shown in FIG. 11(b). No significant difference inviability was observed between DC's exposed to 100 mg of hydrogel orcontrols with no exposure for 24 hours.

Another approach involves pre-encapsulating the biomolecules inmicrospheres, and then embedding these microspheres into the hostscaffold (see FIG. 9(a)). Another approach involves attaching thebiomolecule to the surface of the microsphere. The last approachinvolves gel immobilized hydrogel nanoparticles. These “particle” basedtechnologies are discussed next. The microspheres and nanoparticles arecomplementary technologies (summarized in FIG. 12), both of which are“printable” formulations.

The following discussion provides exemplary methods in which tofabricate the “particles” and how they are incorporated for temporalcontrol of various biomolecules. The first of these controlled-releasecomponents are PLGA/PEG blend microspheres like those shown in theoptical micrograph (OM) of FIG. 12(a). These are prepared by adouble-emulsion technique similar to that reported previously (Ref. 17),and can be used to encapsulate drugs in microspheres having sizestunable from <1 μm to ˜100 μm. PLGA has been used for many years as acontrolled-release material due to its relative biocompatibility andhydrolysis rate. As shown in FIG. 10(b), addition of different amountsof water-soluble PEG in the microspheres allows the release profile ofencapsulated factors to be varied dramatically, due to the formation ofmicroscopic channels in microspheres as PEG dissolves.

The second exemplary component developed for delivery of factors fromthe DCN are biodegradable hydrogel nanoparticles, prepared using acrosslinkable triblock copolymer and a cationic pH-sensitive co-monomer,as illustrated in FIG. 12(b). The nanogel colloid proved miscible withmany of the scaffold materials listed in Table IV. In mixing the nanogelwith collagen, thrombin, and fibrinogen, no significant denaturation ofthe proteins was observed; the fibrinogen/thrombin system completelyretained activity.

These nanogel particles are designed in particular for the delivery ofDNA to cells effectively: (1) encapsulation in the nanoparticles shouldprotect DNA from rapid degradation by extracellular DNAses; (2) theparticles are designed to be readily endocytosed by cells; and (3) theparticles have been engineered to aid the release of DNA into thecytosol by providing a “proton-sponge” effect that can disruptendosomes, triggered by the reduced pH in these intracellularcompartments. The A-B-A triblock is composed of a central PEG B block(4,600 Da) with A blocks composed of PLGA (50:50 w/w lactide:glycolide,each 1,150 Da), and each end of the triblock is capped with amethacrylate group after the approach of Sawhney et al. (Ref. 18).Nanoparticles were synthesized by photopolymerization of awater/oil/water double emulsion. In model DNA delivery experiments, anaqueous solution of pVRC gp120 HIV DNA—250 μL of 0.05 g/mL poly(vinylalcohol) containing 1.6 mg/mL DNA—was added to 1 mL of dichloromethane(Aldrich) containing 200 mg methacrylated PLGA-PEG-PLGA, 350 μL2-diethylaminoethyl methacrylate, and 4 mgphenylbis(2,4,6-trimethylbenzoyl)phosphine oxide photoinitiator. Themixture was sonicated to form an emulsion. This primary emulsion wasthen added to 20 mL of aqueous 0.05 g/mL poly(vinyl alcohol) andsonicated for 30 s to form the second emulsion. The emulsion wassubsequently polymerized by exposing the rapidly stirring solution toultraviolet (365 nm, ˜10 mW/cm²) for 3 minutes. The solution was stirredcontinuously for 2 h to evaporate dichloromethane from the particles.Particles thus obtained were purified by passing through a 0.2-μm filterfollowed by concentration in a 50 kDa centriprep concentrator (Amicon)and separation from free monomer using a PD10 desalting column (AmershamPharmacia). The particles can be fluorescently labeled using rhodaminemethacrylate or fluorescein isothiocyanate methacrylate. The pendantamine groups within the gel particle provide pH sensitivity; thesegroups become charged at reduced pH, causing an electrostatically drivenswelling of gel particles. A scanning electron micrograph (SEM) ofnanoparticles obtained by this process is shown in FIG. 12(c). PlasmidDNA can be encapsulated in these particles, as illustrated by the gelelectrophoresis of DNA recovered from lysed particles (FIG. 12(d)), andthe particles are readily internalized by DC's (FIG. 12(e)). This is animportant finding for the DCN layers, which can be used to deliver DNAplasmids.

As discussed in the previous paragraph, an alternative to usingtraditional molecular factors has been recently introduced. The approachcombines the concepts of gene therapy and bioengineering. Instead ofadministering cytokines or chemokines directly, which leads to majordosing and side-effect issues, it is possible to deliver genes thatencode those molecules to target cells in vivo. The genes are part of aplasmid, a circular piece of DNA constructed for this purpose. Thesurrounding cells (phagocytotic cells such as DC's) take up the DNA andtreat it as their own. They turn into tiny factories, churning out thecytokines (factors) coded for by the plasmid. Because the inserted DNAis “free-floating,” rather than incorporated into the cells' own DNA, iteventually degrades and the factors cease to be synthesized. It has beendemonstrated in animals that 3D biodegradable polymers spiked withplasmids will release that DNA over extended periods and simultaneouslyserve as a scaffold for new tissue formation. The DNA finds its way intoadjacent cells as they migrate into the polymer scaffold, an idea thatwill be tried for the cytokine depot proposed herein. The cells thenexpress the desired proteins/cytokines. This technique makes it possibleto control cytokine release more precisely and over a much longer periodto avoid any possible systemic effects.

These biomolecular delivery approaches may be combined by mixing onefactor with microspheres containing a pre-encapsulated second factor toprovide multiple protein delivery with a distinct release rate for each.The mixed natural or synthetic scaffold and PLGA microspheres willeasily fuse to form a continuous, homogeneous matrix.

Examples of Antigens for Use in DCNs

The DCNs of the invention can be used to treat or prevent infectiousdiseases. One of ordinary skill in the art will understand that the DCNsof the invention can be used to vaccinate subjects against any knowninfectious agent. Examples of infectious agents that cause disease,along with examples of antigens that can be used in the DCN to vaccinateagainst these pathogens, include, but are not limited to: humanimmunodeficiency virus (gp120 protein); malaria (MSP1, AMA1, PfEMP1);tuberculosis (antigen 85 A/B, ESAT-6 and heat shock protein 60);influenza (HA, NA); hepatitis B virus (HBeAg); see, e.g., Letvin N L,Barouch D H, Montefiori D C. Prospects for vaccine protection againstHIV-1 infection and AIDS. Annu Rev Immunol. 2002; 20:73-99; Richie T L,Saul A. Progress and challenges for malaria vaccines; Nature. 2002 Feb.7; 415(6872):694-701; Andersen P. TB vaccines: progress and problems.Trends Immunol. 2001 March; 22(3):160-8.

The DCNs of the invention can also be used to treat or prevent variouscancers, by vaccinating the subject with one or more antigens that willstimulate an immune response against the tumor. Many tumor antigens areknown, and one of ordinary skill in the art will know how to select theappropriate antigen for treating or preventing a specific tumor.Examples of types of cancer and examples of antigens that can be used inthe DCN to vaccinate against these cancers, include, but are not limitedto: melanoma (MART-1, MAGE-1, tyrosinase, gp100, GAGE family); cervicalcancer (human papilloma virus antigens E6 and E7); Burkitt's lymphoma(EBV antigens); CML (bcr-abl fusion product); colorectal, lung, bladder,head and neck (mutant form of p53); B cell non-Hodgkin's lymphoma andmultiple myeloma (Ig idiotype); prostate cancer (PAA, PSA, PSMA);thyroid cancer (thyroglobulin); liver cancer (alpha-fetoprotein); breastand lung (her-2/neu); colorectal, lung, breast (CEA); colorectal,pancreatic, ovarian, lung (muc-1); many cancers (telomerase, oncogenicmutations in RAS, cdk4, p53 or other oncogenes tumor suppressors); see,e.g., Fong L, Engleman E G. Dendritic cells in cancer immunotherapy.Annu Rev Immunol. 2000; 18:245-73.

In addition, one of skill in the art will appreciate that there is alarge number of adjuvants that are known to modulate dendritic cellactivity (e.g. Tlr ligands and cytokines such as IL-2, IL-7, IL-15,IL-13, TNF-alpha, CD40 activators; see Table III). The skilled artisanwill understand that one or more of these modulators can be used in theDCN to stimulate DC maturation for effective anti-pathogen or anti-tumorimmunity. See, e.g., Pardoll D M. Spinning molecular immunology intosuccessful immunotherapy. Nat Rev Immunol. 2002 April; 2(4):227-38.

The Various Layers of an Exemplary DCN Described in Detail

Having discussed the base scaffold biomaterials to construct the DCNlayers, the list of candidate materials used to construct the “capsule”housing the DCN construct for subcutaneous injection, methods to improvethe construction properties of natural polymers, schemes to reduce thedegradation rate of natural polymers, and micro- and nanoparticlestrategies for controlled release of the biomolecules, a detailedexamination is now provided of the individual layers of theheterogeneous DCN ETC and the biomolecules that are embedded in eachlayer to induce a specific response and/or functionality. The digitalprinting BAT can fabricate all the layers of the DCN by depositing themin LBL mode to form a 3D heterogeneous ETC.

First Layer: Monocyte Chemoattractant Layer 0110

The first layer is a monocyte chemoattractant layer 0110 as shown inFIG. 1. This layer attracts monocytes from the blood to the DCN. Thereason for attracting monocytes is that they are a more plentiful cellsource in the blood as opposed to DC's—monocytes comprise approximately30% of the white blood cells, whereas DC's are only about 0.5% of thetotal. The more abundant monocytes make statistical interaction with theDCN more likely.

The monocytes are attracted by a number of chemokines such as fMLP,MIP3-α, and MCP-1, MCP-2, MCP, MIP1α, MIP1β, RANTES, HCC-1, HCC-2,HCC-4, MPIF-1, C5a, b-defensin to name a few. The concentration rangesfor these chemokines are from 1 picomolar tol millimolar (e.g., in thepicomolar and/or micromolar range, e.g., 1-10 pM; 10-100 pM; 100 pM-1μM; 1-10 μM; 10-100 μM, etc.).

Samples have been fabricated to test chemotactic behavior. The Samplebelow is intended to be exemplary only, as one of ordinary skill in theail will understand that many other combinations can be used for thebiomaterial scaffold as well as for the types and combinations ofchemokines. These chemokines can be built into the scaffold matrixduring fabrication, or they are surface-immobilized onnano/microparticles that are added to the scaffold material duringfabrication, or they can be embedded in the nano/microparticles that areadded to the scaffold material during fabrication. For the specificexample provided herein, the samples contain fMLP-O-Me (the methyl esterof fMLP) as the chemoattractant that is embedded in the scaffold matrix,and has been built in LBL mode from fibrin glue components. Generally,the scheme is as such:

The first layer containing fibrinogen or thrombin component (the namesfor the solutions are “Fibro” and “Thrombo,” respectively), is depositedto make a square patch 8×8 mm, ≈200 μm thick.

A solution of fMLP-O-Me is injected deep into the patch in acheckerboard mode (FIG. 14). Multiple short-time injections are madethat cover homogeneously the central 5.1×5.1 mm part of the patch,leaving the margins free (FIG. 15). (These solutions could also beencapsulated in biocompatible microspheres.)

A second 8×8 mm layer of the counter-component, i.e., “Thrombo” if thefirst solution was “Fibro” and vice versa, is deposited to cover thechemokine. Multiple layers can be constructed to make the layers thickerif needed. The samples are left in covered Petri dishes in therefrigerator overnight or over a weekend to dry them out.

The solutions used were:

-   -   (1) “Fibro”: 80 mg/mL human fibrinogen in distilled water+0.3%        HA.    -   (2) “Thrombo”: 22 mg/mL human thrombin in distilled water+0.5%        HA; no Ca²⁺ has been added.    -   (3) fMLP-O-Me: 5 mM in (33% glycerol+67% dimethylsulfoxide,        v/v).

The injection pattern used was:

-   -   162 dots in a shifted checkerboard mode;    -   linear dot-to-dot distance 600 μm;    -   total weight of solution deposited ≈1.2 mg (FIG. 15).        Second Layer: Monocyte Differentiation Layer 0120

The second layer is one that differentiates the more abundant monocytesinto iDC's in the DCN. DC's are the “professional” APC's and hence themost important cell type to the DCN. The biomolecular factors thatinduce differentiation are well known and established in the literature.Several candidates include interferon-α, flt3L, or GM-CSF, IL-4, IL-3,TGFb, IL-15, IL-7, IL-2 proteins as the differentiation factors directlyembedded in the scaffold matrix or surface-immobilized on biocompatiblemicrospheres such as PLGA.

Third Layer: Antigen Presentation Layer 0130

Having differentiated the monocytes to iDCs, the next stage is to loadthe desired antigens into these iDCs. Antigens embedded into thescaffold matrix or surface-immobilized on micro or nano-particles aremethods in which antigen presentation to the iDCs occurs. Such antigenscould be libraries of expressed peptides (1 nanogram-1 milligram; e.g.,10-100 ng; 100 ng-1 μg; 10-100 μg; 100 μg-1 mg, etc.), recombinantpeptides or proteins, DNA plasmids to express antigens, etc. Solidpolymer microspheres for antigen delivery can be composed from suchbiodegradable polymers as PLGA, polyanhydrides, polyphosphazenes, PCL,and their copolymers by single- or double-emulsion fabrication methods.Gel particles can be prepared from biodegradable networks, e.g.,cross-linkable PLGA-PEG-PLGA or PCL-PEG-PCL block copolymers orPEG-peptide-PEG copolymers with an enzymatically degraded peptidesequence (Ref. 19); or nondegradable networks, e.g., ionicallycrosslinked alginate or chitosan, polymethacrylates, or crosslinkeddextrans. Antigens can be encapsulated in gel/solid polymer particles,immobilized to the surface, or both. Antigens engulfed by DC's arereadily loaded onto class II MHC's for presentation to CD4⁺ helper Tcells, but do not load class I MHC's for presentation to CD8⁺ killer Tcells. Because CD8⁺ T cells are likely critical for immune responses topersistent infections and for fighting cancer, the DCN must provide amechanism for loading class I MHC's with chosen antigens. To achievethis, incorporation of micro- and nanogel particles formed using thedegradable triblock copolymers to deliver antigens intracellularly toDC's are employed. These particles, when engulfed by DC's, are designedto disrupt endosomes by swelling at the reduced endosomal pH within DC'sand/or through a “proton sponge” effect (Ref. 20), causing release ofantigen into the cytosol, where it can be loaded onto class I MHC's.

Gel particles encapsulating the model protein antigen ovalbumin havebeen prepared by photopolymerizing an emulsified solution of thetriblock copolymer, protein, and a cationic amino monomer, asillustrated in FIG. 12(b). Initial experiments confirm thatprotein-loaded gel particles are readily taken up by DC's. Shown in FIG.16 are fluorescence/brightfield micrographs from an example DC after 1hour exposure to a nanoparticle suspension. Particles are distributedthroughout the cell body. Fluorescence was stable in cells for severaldays in culture, supporting the hypothesis that these may serve a dualrule as tracers for antigen-exposed cells in vivo. Particle uptake atthe densities shown did not have any acute toxicity for DC's (viabilityequivalent to controls that were not exposed to particles). The maximalprotein antigen loads that can be incorporated in the particles, whatsizes can be prepared, and how degradation rates of the particles can betuned by composition variation are currently being assessed.

The literature provides ample precedent for particle-based class Iantigen loading in DC's. It has been demonstrated (Ref. 21) that antigenadsorbed to the surface of latex beads (and many other types ofparticles) leads to cross-priming and class I antigen loading on DC's.This method of antigen delivery is 100-1000 times more potent thansimply exposing DC's to free protein antigen. However, the fact thatprotein is only adsorbed to particle surfaces is a serious limitation,because only a tiny amount of protein can be delivered. Using thenanoparticles described herein, protein, peptides, or nucleic acid isdistributed throughout the particle volume, allowing potentially1000-fold more Ag to be delivered.

Finally, successful digital printing of nanogels with hyaluronic acidhas been demonstrated, i.e., no agglomeration of the nanogels wasobserved. This shows the demonstration that the antigen presentationlayer of the DCN construct can be easily built.

The ideal vaccine would deliver a simple, low-cost antigenconstitutively to DC's. One way to increase the potency of antigenpresentation would be to use the DCN to transfect in situ DC's with DNAcausing DC's to produce antigen for themselves. This general concept wasdiscussed earlier. To consider this option in our device design, wetested DNA encapsulation with triblock gel particles and found that DNAcan be incorporated similar to proteins. Shown in FIG. 17 (and in FIG.12) is an ethidium-bromide-stained gel electrophoresis result on DNAextracted from nanoparticles, along with DNA standards for comparison.The “unfractionated” lane shows DNA both inside and outside particlesprior to purification, and “fraction 2” shows DNA that was entrapped inparticles (≈40 μg).

In some cases, DNA plasmids may express intracellular antigens forpresentation on MHC class I; in other examples, they may expresssecreted proteins that DCs will carry to and produce in the draininglymph nodes. Secreted proteins may be fusions of DC-binding ligands. Forexample, fusion of Ig or complement C3 with an antigen allows antigensto enter the MHC class I pathway, even when delivered outside of thecell (Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, RescignoM, Saito T, Verbeek S, Bonnerot C, Ricciardi-Castagnoli P, Amigorena S.Fcgamma receptor-mediated induction of dendritic cell maturation andmajor histocompatibility complex class I-restricted antigen presentationafter immune complex internalization. J Exp Med. 1999 Jan. 18;189(2):371-80). In addition, DNA plasmids may express any proteinligands that may modulate dendritic cell maturation for use inparticular disease states (see section below describing layer 4).

Fourth Layer: Maturation and DC Chemoattractant Layer 0140

The fourth layer of the DCN ETC is comprised of a chemoattractant layerto attract IDC's further into the scaffold and of a signal to furthermature the DC's. The DCs are attracted by potentially a number ofchemokines such as fMLP, MIP3-α, and MCP-1, MCP-2, MCP, MIP1α, MIP1β,RANTES, HCC-1, HCC-2, HCC-4, MPIF-1, C5a, b-defensin to name a few(Zlotnik A, Yoshie O. Chemokines: a new classification system and theirrole in immunity. Immunity. 2000 February; 12(2): 121-7; Yang D, ChertovO, Bykovskaia S N, Chen Q, Buffo M J, Shogan J, Anderson M, Schroder JM, Wang J M, Howard O M, Oppenheim J J. Beta-defensins: linking innateand adaptive immunity through dendritic and T cell CCR6. Science. 1999Oct. 15; 286(5439):525-8; Sozzani S, Sallusto F, Luini W, Zhou D,Piemonti L, Allavena P, Van Damme J, Valitutti S, Lanzavecchia A,Mantovani A. Migration of dendritic cells in response to formylpeptides, C5a, and a distinct set of chemokines. J. Immunol. 1995 Oct.1; 155(7):3292-5). The concentration ranges for these chemokines arefrom 1 picomolar to 1 millimolar (e.g., in the picomolar and/ormicromolar range, e.g., 1-10 pM; 10-100 pM; 100 pM-1 μM; 1-10 μM; 10-100μM, etc.). The concentration of the chemokines in this layer will needto be less than that of the monocyte attractant layer (e.g., at least2-fold less; at least 5-10-fold less; at least 10-25-fold less; at least25-50-fold less; at least 50-100-fold less). The lower concentrationcreates an attractive gradient within the DCN to move the DC's throughthe various layers.

Chemokines

Peptide or protein-entrapping microspheres composed of PLGA either aloneor blended with PEG have been tested for controlled release ofchemoattractants in the DCN. These microspheres are formed by a simpledouble- or single-emulsion process (for proteins and peptideencapsulation, respectively) and can be prepared with sizes ranging from<1 μM to >100 μm diameters. By blending different amounts of thehydrophilic polymer PEG with the more hydrophobic PLGA, release profilesfor proteins and peptides from these microspheres can be tailored, asshown in FIG. 10(b).

The formyl peptide fMLP (formyl-Met-Leu-Phe) chemoattractant has beenstudied in addition to the protein chemokine MIP-3α. The formyl peptideis a bacterial byproduct that attracts DC's to sites of infection. Ithas been reported in the literature to be attractive for iDC's in miceand humans. Tests were carried out with this material and found that thepeptide attracted bone marrow-derived dendritic cells with a maximalpotency comparable to MIP-3α (FIG. 18). For these experiments, DC's wereplaced on the top of a migration filter containing 5-μm pores with areservoir of fMLP (or MIP-3α) at the indicated concentration on theother side. After 90 minutes, the number of cells migrating in responseto the chemoattractants was counted and compared to controls. In FIG.18, CI is the chemotaxis index, defined as (number of migrated cells inchemokine)/(number of migrated cells in control without chemokine).

The literature reports CI up to ≈5 max for bone-marrow-derived dendriticcells (BMDC's), but this experiment was carried out on late-stage DCcultures (DC's are starting to mature on Day 7) and the culture was notpurified, thus a significant contamination with neutrophils is likelypresent; thus the real CI is possibly higher. Of importance is that factthat high concentrations of fMLP appear to give comparable results toMIP-3α (which in previous experiments are found gave maximal migrationat 1 μg/mL, in line with literature reports). Having found that fMLPdoes chemoattract DC's, controlled-release PLGA microspheres to deliverthis agent for chemoattraction in the DCN device is the preferredembodiment.

Use of fMLP has numerous advantages over MIP-3α: (1) It is a 3-merpeptide, inexpensive and commercially available in large quantities,hence much more economical both for experiments and from the standpointof viable commercial vaccines; (2) since it is only a peptide, there areno concerns with stability within microspheres/gels or shelf life; and(3) as it is very hydrophobic, it is readily encapsulated in PLGAmicrospheres. (A hydrophobic, low-molecular-weight cargo is the “ideal”case for microsphere encapsulation and release.) PLGA microspheres areused to deliver this agent as its low molecular weight makes itunfeasible to slow its release in hydrogels (it will diffuse outessentially unimpeded).

Maturation Signal

The DC state is an important parameter in determining the nature of theimmune response (Ref. 21). The most basic DC states described in theliterature are the immature and mature states: immature DC's are poisedto capture antigens but lack the requisite accessory signals for T-cellactivation, while mature DC's have a reduced capacity for antigen uptakebut an exceptional capacity for T-cell stimulation. Immature DC's,contrary to previous assumptions, are not ignored by the immune systemand can lead to tolerance by inducing IL-10-producing, antigen-specificregulatory T cells. Maturing DC's redistribute MHC class II molecules tothe plasma membrane and upregulate surface co-stimulatory molecules, MHCclass I, and T cell adhesion molecules. Mature DC's also modify theirprofile of chemokine receptors, which enable homing to lymphoid organs(Ref. 22).

Differences in the expression of MHC, adhesion, costimulatory, and othermolecules as well as differences in cytokine secretion further subdividemature DC states and can influence the nature of the immune response. Ina recent study the different adaptive immune responses produced bylipopolysaccharide (LPS) from different bacteria (Escherichia coli andPorphyromonas gingivalis) were linked to the different cytokineexpression profiles in mature DC's (Ref. 22) (Ref. 22). E. coli LPSinduced a T-helper cell (T_(H)1)-like response, while P. gingivalis LPSinduced a T_(H)2-like response. The DC expression of three cytokines,IL-12, IL-6, and tumor necrosis factor (TNF)-α, was measured. IL-12 wasinduced only in the DC's of E. coli LPS-treated mice; expression of IL-6and TNF-α was similar in DC's from both treatment groups. This findingis consistent with other reports showing that mature, IL-12-producingDC's transform CD4-expressing T-helper cells into IFN-γ-producing T_(H)1cells and lead to cell-mediated immunity, while DC's in the presence ofIL-4 induce T cells to differentiate into T_(H)2 cells and lead tohumoral immunity. Most importantly, understanding the effects ofdifferent DC states allows rational intervention; it is thisunderstanding that is exploited in the DCN. The DCN puts the DC in theright state to activate a desired immune response.

Modulating the Dendritic Cell State

Prior to recent work conducted at the Whitehead Institute (WI), thedownstream target genes induced in DC's by different pathogens had notbeen fully determined. To systemically explore the gene expressionprofile of DC's, WI exposed human-monocyte-derived DC's to a diverse setof organisms and compounds: (1) the Gram-negative bacterium E. coli, andits cell-wall component LPS; (2) the fungus Candida albicans, and itscell-wall-derived mannan; and (3) the RNA virus influenza A, and itsdouble-stranded RNA. DC's were cultured with pathogens or theircomponents and RNA expression was measured using oligonucleotidemicroarrays. FIG. 19 shows an analysis of pathogen-regulated genes aswell as a comparison of mRNA expression levels in response to twopathogens. Image A shows overlapping sets of E. coli, C. albicans, andinfluenza-regulated genes; Image B shows a representation of mRNAexpression levels at 0, 1, 2, 4, 8, 12, and 24 hours in response to E.coli and C. albicans. The colored bars represent the ratio ofhybridization measurements between corresponding time points in thepathogen and control medium profiles.

Of the ≈6,800 genes studied, a total of 1,330 genes changed theirexpression significantly upon encounter with one of the pathogens orcomponents. Such a large-scale change in gene expression demonstratesthat DC's can undergo dramatic transformations in their cellularphenotype. DC maturation, therefore, should not be simply defined by themodulation of a standard set of markers. Table V illustrates the widefunctional variety in genes regulated.

The WI genome-wide analysis of DC gene expression reveals many geneswith potential immunostimulatory roles. For example, anti-apoptoticgenes may extend the lifetime of infected DC's, and matrixmetalloproteases may allow cytokine processing and DC migration to lymphnodes. In addition, many genes with undefined roles in DC function werealso identified, including signaling molecules, transcription factors,and adhesion molecules. Since E. coli differentially up-regulated mostinnate immune response genes on the array, includingneutrophil-attracting chemokines (see Table V), WI tested the in vitromigration of neutrophils toward conditioned cell-cultured mediumcollected from DC's exposed to E. coli, influenza, or control medium. WIfound significant migration with E. coli treatment versus little to nomigration in the influenza or control treatments. Thus, DC statemodulation has consequences for the type of elicited immune response. Itis this DC state modulation that is controlled by the DCN and in partmakes this TE vaccine unique.

The DC states, based on DC gene expression profiles, allow the rationaloptimization of the modulation of DC's for the DCN. Using this knowledgeof DC states and gene expression increases the specificity and potencyof immune responses against pathogens.

Table III displays examples of ligands for use in modulation of DC's inthe biomaterial scaffold for the maturation signals. These signals areembedded in the scaffold matrix, or are surface immobilized onmicrospheres embedded in the scaffold, or are embedded in themicro/nanoparticles that are added to the scaffold. The antigen-loadedDC's encounter the layer that contains these candidate biomolecularstate modulators. In addition, these maturation ligands may also becoupled to antigens covalently or non-covalently. Or, in the case ofprotein ligands, may be fused genetically and expressed as a fusionprotein.

In addition, for pathogens that evade immunity, it may be possible toreverse this evasion with appropriate inhibitors. And finally, in thecase of autoimmune diseases, ligands that are inhibitors of dendriticcell activation will be essential to turn responses toward tolerance; orinhibitors of stimulatory ligands may reduce autoimmunity (such as Tlr9inhibitors: Reference: Leadbetter E A, Rifkin I R, Hohlbaum A M,Beaudette B C, Shlomchik M J, Marshak-Rothstein A. Chromatin-IgGcomplexes activate B cells by dual engagement of IgM and Toll-likereceptors. Nature. 2002 Apr. 11; 416(6881):603-7).

Fifth Layer: Optional Symmetry Layer 0150

The fifth layer is an optional layer largely based on symmetry of theDCN ETC. The fifth layer is not necessary for DCN functionality.However, it may be a comprised of a number of a various material andconstituent formulations, and serve the following optional functionssuch as: (a) a thin scaffold material, with no specific biomolecules, tocontrol the release of the DC's; or (b) an additional antigen-presentinglayer. Thus, if iDC's statistically encounter the DCN, they willphagocytosize the antigens and then encounter the chemoattractant andmaturation signal layers to form fully mature DC's. In this case themotility of the DC's is upward in FIG. 1.

Sixth Layer: Optional Encapsulation Layer

The sixth layer is also optional depending on the releasecharacteristics or the fragility of the DCN ETC. The sixth layer, orreally encapsulating layer, is a biocompatible “capsule” such as thatshown in FIG. 5 and FIG. 6. The encapsulating layer can optionally beloaded with signal molecules (e.g., chemoattractants, antigens, monocyteor DC modulators, etc.)

Variations in Layer Construction of the DCN

The above discussion of the various layers is illustrative only, asseveral of the functionalities of the various layers can be combinedtogether. For instance, layers 0110 and 0120 (monocyte attractant layerand monocyte differentiation layer) are illustrated as distinct layers,but could alternatively be constructed as one layer. The importantaspects of the DCN are what the construct does; it does not necessarilyhave to use distinct layers to accomplish its functionality.

Also, only certain layers of the DCN construct are necessary to inducean enhanced immune response. For example, instead of providing separatelayers for monocyte attraction and differentiation, as described above,one can simply attract iDC's to the construct and load them with chosenantigens and appropriate state modulators. Similarly, it may only benecessary to have an iDC depot to illicit an enhanced immune response.In this case, the only layer required would be the DC chemoattractantlayer 0110. Thus, one of ordinary skill in the art will understand thatvariations, permutations, and combinations of the layers are included inthe present invention.

Constructing the Dendritic Cell Node by Other Means

As described previously, the DCN can be constructed by a LBL depositionprocess using such digital printing processes as that afforded by theBAT. In LBL construction, each layer is subsequently built on top of theprevious layer. However, due to the nature and relative lack ofrestrictions on shape or size of the DCN, it could also be constructed,even in layered fashion, through other methods. Two examples areillustrated below.

Folded Constructs

One alternative method by which to construct a layered DCN is to“sandwich” the membranes. Specifically, a TE membrane biomaterial couldbe designed to include various individually engineered borders orsections, e.g., quadrants. In each border or section, the appropriatevarious biomolecular factors are added, then the whole structure isfolded so as to create a 3D structure, as shown in FIG. 20. For example,in a four-quadrant folded structure, biocompatible chemokinemicrospheres could be placed in the upper-right quadrant II, nanogelscontaining DNA plasmids could be placed in the lower-right quadrant III,and structural materials could be placed in quadrants I and IV, whichbecome the outermost layers.

These engineered quadrants could be constructed in a number of ways byusing the BAT, such other digital printing tools as electrosprays andinkjets, or such manual printing tools as micropipets. After thequadrants are constructed, the membrane is then folded in such a waythat the various layers are still distinct and in the proper order fromthe topmost to the bottommost layers. In FIG. 20, this is accomplishedby folding the originally flat xy-plane structure around the y axis,then by folding the resultant yz-plane structure around the x axis. (Inthe figure, thickness is exaggerated to show the layered structure.) Forthis to be possible, the membrane must be thin, pliable, and flexible,besides biocompatible. Candidates for such membranes could include ECMsheets, fibrin sheets, or collagen sponge scaffolds.

Roll-to-Roll Constructs

Another method by which to fabricate a DCN in layered fashion is to usea roll-to-roll process in essence comparable to the web-handlingtechniques widely used in printing and other industries. The basicscaffold or substrate material should be thin, pliable, and flexible yetbiocompatible; suitable materials include ECM, fibrin, or collagen.

The advantage of modern computer-controlled web-handling techniques isthat the substrate sheet moves from the feed or input roll to the uptakeor output roll at a known rate. Such parameters as the angularvelocities of the two rolls and the resultant thickness of the layersdeposited onto the output roll can be calculated and controlled.Meanwhile, the motion of the sheet past the writing heads and tabledetermines the rate at which the active components of the DCN must bedeposited.

As the substrate moves past, various dispensing units, such aselectrosprays, inkjets, BAT printing elements, micropipets, or othertools can be used to “print” the various biomolecular components ontothe substrate in conveyor-belt fashion. Once these printed regions reachthe output roll, the individual printed layers can be compiled to makethe overall 3D structure with the separate layers still resolved, whichin this case will have cylindrical symmetry, as is illustrated in FIG.21.

Immune Modulation by the DCN

In one specific example, the present invention provides a method bywhich DCN-hosted DC's offer a solution to the previous problem ofdeveloping a malaria vaccine that can initiate T-cell responses at onestage and B-cell responses at others. It is now apparent that the key toan effective malaria vaccine is that it must initiate both T_(H)1 andT_(H)2 responses, leading to the stimulation of cytotoxic T lymphocytes(CTL's) and antibody-producing B lymphocytes. Previous vaccine researchhas focused upon only one of these pathways, T_(H)1 producing CTL's orT_(H)2 producing antibodies. Existing vaccines do not work well becauseof this limitation of focus and temporal control. The DCN is the onlypresent technology that allows the initiation of T_(H)1 responses atcertain stages and T_(H)2 at others. Ordinarily, the T_(H)1 and T_(H)2pathways cannot be induced simultaneously by a single conventionalvaccine because the T_(H)1 cytokines block the T_(H)2 pathway and viceversa. However, the novel aspect of the DCN operates by making itpossible to induce these different immune responses at different times,on demand. The DCN can also be used to modulate DCs to block the T_(H)2pathway, thereby blocking allergic responses.

The way the type of immune response can be controlled via the DCN ETC isby controlling the degradation rates of the scaffold material and themeans of its construction via a layer-by-layer growth mechanism. Forexample, some of the ETC layers could be built to have largely a T-cellresponse (e.g., by incorporating IL-12, IL-2, or IFN-γ in the scaffoldmatrix during fabrication) followed by layers that would induce a B-cellresponse (e.g., by incorporating IL-4 and IL-10 in the TE scaffoldduring fabrication), etc.

For autoimmune diseases, it is possible to construct an DCN withantigens that are found as targets of autoimmune responses (e.g. insulinor GAD for diabetes, myelin basic protein for multiple sclerosis,acetylcholine receptor for myasthenia gravis, etc.) and state modulatorsthat would turn dendritic cells into tolerizing cells (e.g. vitamin D,IL-10 or other tolerizing agents), thus leading to the reduction of theautoimmune response due to T and B cells. (Yoon J W, Jun H S. Cellularand molecular pathogenic mechanisms of insulin-dependent diabetesmellitus. Ann N Y Acad. Sci. 2001 April; 928:200-11; MS, Stinissen P,Medaer R, Raus J. Myelin reactive T cells in the autoimmune pathogenesisof multiple sclerosis. Mult Scler. 1998 June; 4(3):203-11; De Baets M,Stassen M H. The role of antibodies in myasthenia gravis. J Neurol Sci.2002 Oct. 15; 202(1-2):5-11; S. Gregori, N. Giarratana, S. Smiroldo, M.Uskokovic, and L. Adorini A 1 {alpha}, 25-Dihydroxyvitamin D3 AnalogEnhances Regulatory T-Cells and Arrests Autoimmune Diabetes in NOD MiceDiabetes, May 1, 2002; 51(5): 1367-1374; M. D. Griffin, W. Lutz, V. A.Phan, L. A. Bachman, D. J. McKean, and R. Kumar Dendritic cellmodulation by 1alpha, 25 dihydroxyvitamin D3 and its analogs: A vitaminD receptor-dependent pathway that promotes a persistent state ofimmaturity in vitro and in vivo. PNAS, Jun. 5, 2001; 98(12): 6800-6805).

Other examples of tolerizing agents that can be used in the DCN includeaspirin, steroidal or non-steroidal anti-inflammatories, ATP, TGF-β,ligands or activators of the following receptors: SIR-P, CD36, mer orDC-SIGN; as well as several other ligands shown in Table III(troglitazone, bradykinin, etc). Alternatively, by ensuring that DCsattracted to the DCN are immature (i.e. by not providing any activatorsin the DCN construct), tolerance will ensue. Finally, by attractingplasmacytoid DCs specifically, it should be possible to induce tolerancewith or without a maturation-inducing stimulus in the DCN. In summary,there are many ways to block dendritic cell maturation and ensure that Tand B cells are not optimally activated and undergo tolerance (anergy,deletion or differentiation into regulatory T cells) instead ofactivation. (See, e.g., Yin et al. The anti-inflammatory agents aspirinand salicylate inhibit the activity of IkB kinase-beta. Nature 1998,396:77; Webster et al. Neuroendocrine Regulation of Immunity. Annu. Rev.Immunol. 2002, 20:125-63; la Sala et al. Extracellular ATP Induces aDistorted Maturation of Dendritic Cells and Inhibits Their Capacity toInitiate Th1 Responses. J. Immunol., 2001, 166: 1611-1617; Latour et al.Bidirectional negative regulation of human T and dendritic cells by CD47and its cognate receptor signal-regulator protein-alpha: down-regulationof IL-12 responsiveness and inhibition of dendritic cell activation. JImmunol 2001 Sep. 1; 167(5):2547-54; Britta et al. A role for CD36 inthe regulation of dendritic cell function. PNAS 2001 vol. 98(15):8750-8755; Cohen et al. Delayed Apoptotic Cell Clearance and Lupus-likeAutoimmunity in Mice Lacking the c-mer Membrane Tyrosine Kinase. J. Exp.Med 2002 Volume 196, Number 1, Jul. 1, 2002 135-140; Teunis et al.Mycobacteria Target DC-SIGN to Suppress Dendritic Cell Function. J. Exp.Med. 2003 Volume 197, Number 1, Jan. 6, 2003

7-17; Dhodapkar and Steinman. Antigen-bearing immature dendritic cellsinduce peptide-specific CD8(+) regulatory T cells in vivo in humans.Blood 2002 Jul. 1; 100(1):174-7; Gilliet and Liu. Generation of humanCD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendriticcells. J Exp Med 2002 Mar. 18; 195(6):695-704).

Exemplary DCN Constructions

The following provides examples of combinations of monocyte chemokines,differentiation proteins, antigens, maturation ligands, andchemoatttrants that can be used to construct the DCNs of the invention.These examples are not intended to be limiting, as it will be clear toone of ordinary skill in the art that any appropriate combination ofmonocyte chemokines, differentiation proteins, antigens, maturationligands, and chemoatttrants as described herein or as known in the artor later discovered can be used to construct the DCNs of the invention.

Components of a DCN for Treating or Preventing an HIV Infection

-   1. Monocyte chemokine layer: fMLP, and/or MIP3α, to attract    monocytes from the blood to the DCN.-   2. Monocyte differentation protein layer: flt3L, INF-α to    differentiate monocytes into dendritic cells.-   3. Antigen layer: either recombinant gp120 protein (Genbank    NC_(—)001802) or a DNA plasmid version with gp120 fused to the Fc    portion of human Ig in order to get efficient B cell responses as    well as T cell responses (gp120-Fc fusion will bind to the    follicular dendritic cells that present antigens to B cells and    stimulate B cells antibody production).-   4. Maturation layer ligands and chemoattractant: CpG oligo for the    ligand, and fMLP or MIP3α for the chemokine. The chemokine    concentration of this layer should be less than that of layer 1 (at    least two-fold less).-   5. Antigen layer: same as 3.    Components of a DCN for Treating or Preventing Diabetes-   1. Monocyte chemokine layer: MIP3α.-   2. Monocyte differentation protein layer: flt3L.-   3. Antigen layer: insulin-B (Genbank Accession No. J00265) or GAD    (Genbank Accession No. M74826).-   4. Maturation layer ligands and chemoattractant: Vitamin D or IL-10    for the ligands, and MIP3α for the chemokine. The chemokine    concentration of this layer should be less than that of layer 1    (e.g., at least two-fold less).-   5. Antigen layer: same as 3.    Components of a DCN for Treating or Preventing Multiple Sclerosis-   1. Monocyte chemokine layer: MIP3α.-   2. Monocyte differentation protein layer: flt3L.-   3. Antigen layer: myelin basic protein (Genbank Accession No.    X17286).-   4. Maturation layer ligands and chemoattractant: Vitamin D or IL-10    for the ligands, and MIP3α for the chemokine. The chemokine    concentration of this layer should be less than that of layer 1    (e.g., at least two-fold less).-   5. Antigen layer: same as 3.    Components of a DCN for Treating or Preventing Myasthenia Gravis-   1. Monocyte chemokine layer: MIP3α.-   2. Monocyte differentation protein layer: flt3L.-   3. Antigen layer: acetylcholine receptor alpha subunit (Genbank    Accession No. y00762).-   4. Maturation layer ligands and chemoattractant: Vitamin D or IL-10    for the ligands, and MIP3α for the chemokine. The chemokine    concentration of this layer should be less than that of layer 1    (e.g., at least two-fold less).-   5. Antigen layer: same as 3.    Advantages of the DCN

Use of an ETC to harbor chemokines, cytokines, modulators, and/orantigens for the DCN, with or without exogenously-added DC's, provides ahub to attract and “train” DC's to present a chosen antigen, as well asa biocompatible harboring site designed to keep the DC's alive. The DCNprovides the proper microenvironment/spatial control to modulate andprogram the DC's to induce a specific immune response. Moreover, thebiodegradable natures of the scaffold and the embedded biomolecules,microspheres, or nanoparticles containing the biomolecules providetemporal control over any specific arm of the immune system and/orrelease of specific cytokines or chemoattractants.

Use of the DCN to stimulate or tolerize the immune system has numerousadvantages, as has been discussed herein. For example, the DCNconcentrates DCs by attracting them to a small volume in the body (e.g.subcutaneously), and enhances antigen delivery to DCs by providing largeamounts of antigen where DCs are attracted and concentrated. The DCNalso enhances DNA plasmid or viral-based delivery of antigens byconcentrating DCs and thus effectively increasing specific delivery ofDNA and viral particles to DCs rather than other cell types (e.g.fibroblasts, endothelial cells, muscle cells, keratinocytes). Moreover,use of nanoparticles for antigen presentation greatly enhances theamount of antigen that is presented to the DCs.

In addition, the DCN modulates the state of concentrated dendritic cellsuniformly using protein or non-protein ligands (including smallmolecules) that regulate the activity of specific receptors or proteinsexpressed in dendritic cells. Moreover, the DCN can employ DNA vaccinesor viral vectors to express genes that can modulate the DC state.

DCNs can contain bioconcrete in any or all layers, to reduce thedegradation rate of biomaterials within the DCN. The bioconcrete cancontain bioactive substances, such as (but not limited to) chemicals,peptides or polypeptides, anti-virals, for controlled drug release. Thebioconcrete can also contain microspheres and/or nanoparticlescontaining such bioactive substances.

Incorporation by Reference

Throughout this application, various publications, patents, and/orpatent applications are referenced in order to more fully describe thestate of the art to which this invention pertains. The disclosures ofthese publications, patents, and/or patent applications are hereinincorporated by reference in their entireties, and for the subjectmatter for which they are specifically referenced in the same or a priorsentence, to the same extent as if each independent publication, patent,and/or patent application was specifically and individually indicated tobe incorporated by reference.

OTHER EMBODIMENTS

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

LITERATURE CITED

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21. I. Mellman and R. M. Steinman, “Dendritic Cells: Specialized andRegulated Antigen Processing Machines,” Cell 2001, 106 (3), 255. TABLE IAbbreviations and Symbols DCN Dendritic Cell Node nD n-Dimensional (n =3) APC Antigen-Presenting Cells NKC Natural Killer Cell BAT BiologicalArchitectural Tool NMR Nuclear Magnetic Resonance BMDCBone-Marrow-Derived Dendritic Cell OM Optical Micrograph/scope BWBiological Warfare PBS Phosphate-Buffered Saline CAD Computer-AidedDesign PCL Poly(caprolactone) CAM Computer-Aided Manufacturing PEGPoly(ethylene glycol) CI Chemotaxis Index PF-127 Pluronic F-127 CTLCytotoxic T Lymphocyte PLCL Poly(lactide-co-caprolactone) DC DendriticCell PLGA Poly(lactide-co-glycolide) DNA Deoxyribonucleic Acid PPFPoly(propylene fumarate) ECM Extracellular Matrix PPTD(PLGA-co-PEG)-triblock-dimethacrylate ETC Engineered Tissue ConstructSEM Scanning Electron Micrograph/scope HA Hyaluronic Acid TETissue-Engineered HIV Human Immunodeficiency Virus T_(H)1 Helper T Cell,Type I iDC Immature Dendritic Cell T_(H)2 Helper T Cell, Type II IFNInterferon Tlr Toll-Like Receptor LBL Layer-by-Layer T_(m) Temperature,Melting Point (° C.) LPS Lipopolysaccharide UBM Urinary Bladder MucosaMHC Major Histocompatibility Complex WI Whitehead Institute MITMassachusetts Institute of Technology x, y, z Cartesian Coordinates (m)

TABLE II Two Modes of the DCN Scaffolds without Cells Scaffolds withCells Load scaffold with: 1) Start from dendritic cells pulsed 1)chemokines/cytokines; with desired antigens or modulators. 2) targetantigens; 2) Load scaffolds with cells and 3) modulators for desiredimmune cytokines. response (immunity or tolerance).

TABLE III Examples of Dendritic Cell Modulators Receptor ModulatorFunctional Category Serotonin Serotonin Neurotransmitter Receptor β1GABA A GABA Neurotransmitter Receptor β2 Bradykinin Receptor BradykininPain/Inflammation Somatostatin Somatostatin Neuropeptide Receptor 5Vasopressin Vasopressin Vasopressor Peptide Receptor 1B PPARγ Receptor15dPGJ(2), Troglitazone Endocrine Regulation FK506 Binding FK506Immunomodulator Protein Vitamin D Receptor Vitamin D — Purine ReceptorATP, adenosine Endogenous ligand P2x4 TGFb Receptor II TGFb CytokineIL-2R IL-2 Cytokine IL-4R IL-4 Cytokine IL-7R IL-7 Cytokine IL-13Ra1IL-13 Cytokine IL-15 IL-15 Cytokine 4-1BB 4-1BB Ligand ImmunomodulatorCD40 CD40 ligand Immunomodulator RANK RANK ligand Modulator of DCsurvival Tlr-4 LPS, hsp6, hsp70, Pathogen Component hyaluronic acidfragment, saturated fatty acids Tlr-3 dsRNA (e.g. polyI:C) PathogenComponent Tlr-9 CpG DNA Pathogen Component Tlr-2 Bacterial Lipoproteins,Pathogen Component hsp60, SP-A, peptidoglycan, GPI anchor from T. cruzi.Tlr-5 Flagellin Pathogen Component Tlr-1 Mycobacterial lipoproteinPathogen Component triacylated lipopeptides Tlr-6 Mycobacterial PathogenComponent lipoproteins, lipoteichoic acid, peptidoglycan Tlr-8Resiquikopd, imiquimod Synthetic compounds

TABLE IV Examples of Scaffold Materials Material Summary RatingPoly(propylene fumarate-co- PPF-PEG still represents the bestcombination of *** ethylene glycol) (PPF-PEG) constructive properties.PEG-diacrylates and These produce weak hydrogels that are improper forcell * -dimethacrylates attachment. They can be used for cytokine orantigen delivery. Poly(ethylene oxide) Poly(ethylene oxide) gels aresoluble but have minimal * constructive properties.Poly-4-hydroxybutyrate This strong, natural, biodegradable plastic has avery high * melting point, T_(m) > 175° C. Poly(ethylene-co-vinylacetate) This strong, non-biodegradable plastic has T_(m) ≧ 100° C. *(PLGA-co-PEG)-triblock- PPTD is “friendly” to cells. Its constructiveproperties are. *** dimethacrylate (PPTD) fair, weaker than PPF-PEG.Pluronic F-127 (PF-127) This is an excellent shape-former due toinverse-temperature ** gelation, and it is a good drug carrier. Combinedwith PPF- PEG, it allows building real 3D constructs. ExtracellularMatrix (ECM), These are promising cell-carriers, entirely natural and*** Small Intestine Submucosa, and biodegradable. They requireadditional processing Urinary Bladder Mucosa (UBM) (homogenization andcombination). Collagen Type I Produces weak and partially solublehydrogels; it is cell- ** friendly. Calfskin Gelatin This is a goodconstruction material when deposited hot. It is ** cell-friendly andsoluble. Fibrinogen, Thrombin These form insoluble, stable,biodegradable, and cell-friendly *** hydrogel clots. Hyaluronic Acid(HA) This universal biological thickener is cell-friendly. It can be ***safely combined with any other member of this table. Poly(caprolactone)(PCL), Common biodegradable polyesters used extensively in tissue ***Poly(lactide-co-caprolactone) engineering applications; require printingfrom organic (PLCL), poly(D.L-lactide) solvent but provide improvedmechanical strength to the construct. Photocurable Gelatin Astyrene-derivitized gelatin, this was combined with a ** water-solublecarboxylated camphorquinone as a photoactivator. The material has provento be a promising biodegradable and biosorbable hydrogel, whichadhesiveness to living tissues is sometimes superior to that of fibringlue. Polyphosphazenes Biodegradable synthetic polymers that degrade toneutral byproducts. Trimethylene Carbonate Tough, slowly degradingpolymers with good structural Copolymers properties. Scaffold AdditivesThese are not scaffolds per se, but are components of *** (Laminin andFibronectin) extracellular matrix materials that may be necessary forcell proliferation and viability.

TABLE V Functional Categories of Genes Differentially RegulatedNeutrophil GAN EC CA IA INNATE i18 Y00787 +++ + + gro1 X54489 +++ + ±gro2 M57731 +++ + ± gro3 X53800 ++ ± ± Inflammation infa X02910 ++ + +il1b X04500 +++ + + i16 X04602 ++ + + il1a M28983 + − − gcsf X03656 ++ −− mip1b M69203 ++ + ++ mip3a/larc U64197 ++ ± ± mip3b/elc U77180 + ± ±bf L15702 ++ + ± Prostaglandin/Leukotrienc ptgir D38128 ++ + + ptger4L28175 − − + cox2 U04636 ++ + + ADAPTIVE T Cell-Th1 il12b/p40 M65290++ + − itac U59286 + + ++ mig X72755 + + ++ inp10 X02530 + + ++ ifnb1V00535 + − ++ ifna2 J00207 − − + ifna13 J00210 − − +++ ifna14 V00533 −− + ifna16 M28585 − − + T Cell-Th2 tarc D43767 ++ + ± mdc U83171 + + ± TCell Stimulation 41bbL U03398 ++ − ± slam U33017 +++ + + cd86U04343 + + + icam1 M24283 ++ + ++ ebi3 L08187 ++ + − AntigenPresentation b2m J00105 ++ − ++ Lmp10 X71874 + + ± B cell pbef U02020+++ + + IMMUNE RECEPTOR il15ra U316284 ++ ± + il7r M29696 ++ + + il2rX01057 + ± − il4r X52425 + + ± gmcsfr X17648 + ± − il3r D49410 + + −41bb U03397 +++ ± ++ infr2 M32315 ++ − − il13ra1 Y10659 ++ ++ − Cd155M24406 +++ − − Cd83 Z11697 ++ ++ ++ IMMUNE TRANSCRIPTION nfkb p52 S76638++ ± + nfkbp50 M58603 ++ ± ++ nfkb p65 L19067 + + + nfkb re1B M83221 + ±− stat5a U43185 ++ + − stat4 L78440 ++ + − stat3 L29277 + − ± irf2X15949 + − + irf4 U52682 + ± ± isgf3 M87503 + ± + csda M24069 ++ − −GLYCOLYSIS AND ENERGY enol M14328 + − d Pk3 X56494 ++ − ± Tpi J04603 + −− gys J04501 + − d pgm1 M83088 + ± d Gk X69886 + ± − pfkp D25328 + − −pgk1 V00572 + − ± g3pdh X01677 + − ± Ldh1 X02152 + − ± pgd U30255 + − ±pgam1 J04173 + + + Hifla U22431 + − ± APOPTOSIS Inhibitor Pai2 M31551 ++− − Iex-1 S81914 ++ + − Tax1bp1 U33821 + − ± Flip AF005775 ++ + ± bag1Z35491 + + ± ciap2 U37546 ++ ++ + Bc12-a1 U29680 ++ + + mc11 L08246 +− + Tau X56468 ± ± ++ Activator casp4 U28014 ++ + ± Nip3 U15174 ++ ± −trail U37518 + + ++ Fas X63717 + + + casp5 U28015 + − + bak1 U16811 ±± + pmaip1 D90070 + − ++ casp10 U60519 ± + + GROWTH FACTORS ANDRECEPTORS tgfa X70340 + − − ndp X65724 +++ − − wnt5a L20861 +++ ± −aclivinba X57579 +++ ++ + p2x4 AF000234 + + − vdr J03258 + ± − TISSUEREMODELING mmp9 J05070 ± + − mmp7 L22524 ++ − − mmp3 X05232 + − − mmp19X92521 + ± ± mmp14 Z48481 ++ − ± mmp12 L23808 ++ + − mmp10 X07820 + − −mmp1 X54925 + − − lad1 U42408 + − ± cxt12 U76189 ± + ± collagen-M55998 + − − a1 tnr X98085 + − ± CELL STRESS mt1g J03910 +++ ± ± mt1cM10942 +++ ± ± btg2 U72649 ++ ± − fth1 L20941 ++ + − quiescin L42379 ++± − cagb M26311 ++ − ± ddit1 M60974 ++ − ± map3k4 D86968 ++ ± ± mt1lX76717 ++ − ± mt1h X64177 ++ + + mt2a V00594 ++ + + hspa1a M11717 ++ ±++ ninj1 U72661 ++ + ++ sod2 X07834 ++ + ++ atox1 U70660 + + − hspa6X51757 + ± − krs1 U26424 + ± − mt1a K01383 ++ − − mt1f M10943 + − − rtpD87953 ++ ± − cyp450db1 X07619 + ± ± gst12 U46499 + − ± hsf4 D87673 + −± hspa4 L12723 + − ± dusp1 X68277 + − + mtf X78710 + − + hsp70 U10284 ±− + hsp27 Z23090 − ± +++ cbr1 J04056 ± + + IMMUNE INHIBITORS mcp1S69738 + ++ ++ il10 U16720 ++ − − hla-e X56841 − − + gfrp U78190 d − +ido M34455 ++ ± ++ Table Abbreviations and codes + Gene expression isup-regulated in response to pathogen. − Gene expression is not changed.++, Gene expression is changed at a higher level relative to other +++pathogens that regulate the same gene (each + denotes increasedexpression by a factor of ˜2.5). ± Gene expression is regulated in asubset of donors. d Gene expression is down-regulated. GAN GenbankAccount Number EC Escherichia coli CA Candida albicans IA Influenza Avirus

1. A dendritic cell node comprising: a) a biocompatible scaffold material; b) a chemokine for attracting immature dendritic cells; c) a chosen antigen; and d) a maturation signal for dendritic cells. 2-25. (canceled)
 26. A method of constructing a dendritic cell node comprising: a) depositing, onto a substrate, a layer for attracting monocytes into the dendritic cell node; b) depositing, onto layer (a), a layer for inducing differentiation of the monocytes into immature dendritic cells; c) depositing, onto layer (b), a layer for presenting a chosen antigen to immature dendritic cells; d) depositing, onto layer (c), a layer for attracting dendritic cells and inducing maturation of dendritic cells, thereby constructing a dendritic cell node.
 27. (canceled)
 28. A method of constructing a dendritic cell node comprising: a) depositing, onto a substrate, a layer for attracting immature dendritic cells into the dendritic cell node; b) depositing, onto layer (a), a layer for presenting a chosen antigen to the immature dendritic cells; and c) depositing, onto layer (b), a layer for attracting immature dendritic cells and inducing maturation of the immature dendritic cells; or d) depositing, onto a substrate, a layer for attracting immature dendritic cells and inducing maturation of the immature dendritic cells; e) depositing, onto layer (d), a layer for presenting a chosen antigen to the immature dendritic cells; and f) depositing, onto layer (e), a layer for attracting immature dendritic cells into the dendritic cell node, thereby constructing an dendritic cell node. 29-47. (canceled) 